Laboratory detection of COVID-19 using Real Time RT-PCR

COVID-19 is an infectious disease caused by severe acute respiratory syndrome corona virus two(SARS-COV2). when the person is infected the most common sign include fever, cough, shortness of breath.
According to the World Health Organization (WHO), the WHO China Country Office was informed of cases of pneumonia of unknown etiology in Wuhan City, Hubei Province, on 31 December 2019. A novel coronavirus currently termed 2019-nCoV was officially announced as the causative agent by Chinese authorities on 7 January. 

What is real time RT-PCR

Real time Reverse Transcriptase Polymerase Chain reaction(real time RT–PCR) is a nuclear-derived method for detecting the presence of specific genetic material in any pathogen, including a virus. Originally, the method used radioactive isotope markers to detect targeted genetic materials, but subsequent refining has led to the replacement of isotopic labelling with special markers, most frequently fluorescent dyes. This technique allows scientists to see the results almost immediately while the process is still ongoing, whereas conventional RT–PCR only provides results at the end of the process.

Real time RT–PCR is one of the most widely used laboratory methods for detecting the COVID-19 virus. While many countries have used real time RT–PCR for diagnosing other diseases, such as Ebola virus and Zika virus, many need support in adapting this method for the COVID-19 virus, as well as in increasing their national testing capacities.

How real time RT PCR works in COVID-19 detection

The sample is collected by nasal pharyngeal swab or oral pharyngeal swab, for nasal pharyngeal  the swab is inserted in the nostril and gently moved forward in the nasal pharynx and then rotated for a specified period of time to collect secretion containing the virus. then the swab is placed immediately in the sterile tube containing the viral transport media.

Because corona virus contain extra ordinary single stranded RNA genome, to detect this virus with PCR, RNA molecule must be converted into complementary DNA by reverse transcriptase. the obtained DNA is then amplified by real time RT-PCR, The sample is treated with several chemical solutions that remove substances such as proteins and fats and that extract only the RNA present in the sample. This extracted RNA is a mix of the person’s own genetic material and, if present, the virus’s RNA.

The RNA is reverse transcribed to DNA using a specific enzyme. then additional short fragments of DNA that are complementary to specific parts of the transcribed viral DNA are added. If the virus is present in a sample, these fragments attach themselves to target sections of the viral DNA. Some of the added genetic fragments are used for building DNA strands during amplification, while the others are used for building the DNA and adding marker labels to the strands, which are then used to detect the virus.

The mixture is then placed in an RT–PCR machine. The machine cycles through temperatures that heat and cool the mixture to trigger specific chemical reactions that create new, identical copies of the target sections of viral DNA. The cycle is repeated over and over to continue copying the target sections of viral DNA. Each cycle doubles the previous number: two copies become four, four copies become eight, and so on. A standard real time RT–PCR set-up usually goes through 35 cycles, which means that, by the end of the process, around 35 billion new copies of the sections of viral DNA are created from each strand of the virus present in the sample.

As new copies of the viral DNA sections are built, the marker labels attach to the DNA strands and then release a fluorescent dye, which is measured by the machine’s computer and presented in real time on the screen. The computer tracks the amount of fluorescence in the sample after each cycle. When a certain level of fluorescence is surpassed, this confirms that the virus is present. Scientists also monitor how many cycles it takes to reach this level in order to estimate the severity of the infection: the fewer the cycles, the more severe the viral infection is.